| Periodontal disease is an infection of periodontal tissue. According to previous statistics, 2 / 3 of the Chinese population suffer from periodontal disease, which is the main reason of tooth loss of adults over 40 years. The supporting tissue, which includes gum, cementum, periodontal membrane, alveolar bone, is the main affected area. The symptoms of periodontal disease vary from bone absorption, attachment loss, tooth mobility or displacement, pain, inability to chew and final tooth loss. The goal of periodontal therapy is to remove the periodontal pathogenic factors to prevent further attachment loss and to obtain periodontal soft and hard tissue regeneration. Regeneration of alveolar bone is the most critical part of the treatment. Guided tissue regeneration is currently widely used in clinical treatment of periodontal disease. Although to some extent it is effective, it is not fully able to repair and regenerate the affected tissue.Achieving effective periodontal regeneration is the aim researchers have been pursuing. Tissue engineering, which includes the source of seed cells, bio-carrier stent choice and micro-environment construction, has good potential. Optimization of seeding cells is among the most important content of tissue engineering on periodontal regeneration.Through the directional migration and differentiation of different cell subsets in the periodontal ligament, periodontal tissue has a certain self-repair capability. In recent years, using enzyme digestion-forming cells in single cell suspension from periodontal ligament of healthy adults, Seo BM, etc. isolated cells with cloning capability and a high degree of proliferation, named as periodontal ligament stem cells. This provides a new method for the treatment of periodontal disease. Periodontal ligament connects the tooth and alveolar bone. During tooth extraction, periodontal ligament ruptures, and there were residual periodontal ligament stem cells on the teeth and in the alveolar fossa. So the question if that, after tooth extraction, whether the root surface periodontal ligament stem cells and alveolar fossa residual periodontal ligament stem cells, is the same? Do they play a same role in periodontal regeneration? When selecting the seed cell for tissue engineering on periodontal regeneration, which periodontal ligament stem cell is more effective? Studying and answering these questions will undoubtedly contribute to the theoretical foundation of seed cell selection for periodontal tissue regeneration and further promote the effectiveness of tissue engineering treatment of periodontal diseases.The study is divided into two parts, in vitro identification of the socket-derived periodontal ligament stem cells, and an animal experimental study on the in vivo osteogenic potential of this cellThe main contents and results are as follows:1, the osteogenic ability of human periodontal ligament stem cell of different source in nude miceThe first step is the separation and culture of PDLSCs of two different sources. a-PDLSCs (alveolar bone surface periodontal ligament stem cells) is obtained through scraping the residue periodontal membrane in the socket after tooth, and the tissue of enzyme digestion. r-PDLSCs (root surface periodontal ligament stem cells) is obtained through the method described in literature. After indirect immuno beads purified these two PDLSCs. We used osteogenic inducing medium for induction in vitro mineralization. Two weeks later the formation of mineralized nodules were observed in both cells. Through the formation of alizarin red stained mineralized nodules quantitative analysis, we found the area a-PDLSCs in vitro mineralized nodule was larger than the area of r-PDLSCs mineralized nodules in vitro, a-PDLSCs mineralized nodule area is 8.54 %, while the r-PDLSCs mineralized nodule area is 3.21%, indicating a-PDLSCs induced osteogenic potential in vitro was stronger than r-PDLSCs.Two PDLSCs were combined respectively with CBB, and transplanted subcutaneously into nude mice to observe for 6 weeks. We found that a typical periodontal ligament / cementum complex-like structure was formed in r-PDLSCs group; while bone-like mineralized tissue with bone cells was observed in the a-PDLSCs group.So we can deduce that although both cells have the same origin, there is a difference in bone inducing capability in vitro between 2 PDLSCs. a-PDLSCs showed stronger osteogenic induction in vitro ability; and in vivo, a-PDLSCs also demonstrated stronger osteogenic potential.2. The repair ability of defects of skull in nude mice of two kinds of cellOn the basis of the first part of the experiment, we now have purified a-PDLSCs and r-PDLSCs. Through the use of immune-deficient mice, we established the skull defect model with a diameter of 6mm. with fibrin glue as a scaffold material, respectively two cells combined and implanted in nude mice skull defect site. A fibrin glue without any seed cell compounded was used as a blank control group. Micro-CT filming after 0 weeks, 5 weeks and 10 weeks and the HE biopsy tissue after 10 weeks were observed.After 5 weeks, r-PDLSCs and a-PDLSCs skull defect have varying degrees of repair, with an area of about 19%, and 32%respectively, while the control group almost had no new bone formation. After 10 weeks, r-PDLSCs and a-PDLSCs group had a larger area of repair than five weeks, a-PDLSCs group up to 50%and the r-PDLSCs group 35%. A high-density new bone at the defect edges and the middle part of the region also appeared, which was closer to normal newborn bone mineral density.After 10 weeks, through HE slice observation, we found that r-PDLSCs and a-PDLSCs group have a large area of new bone formation, osteoblasts are visible, while the control group almost no new bone formation.In this study, r-PDLSCs showed its new bone induction ability, but a-PDLSCs have a stronger ability of new bone formation, and faster repair of skull defect. So it is an ideal seed cells for bone tissue engineering.3. Periodontal ligament stem cell-mediated treatment of periodontal defects in different parts of beagleThe ultimate goal of periodontal therapy is to achieve complete periodontal tissue regeneration. In this study, we produced a periodontal defects model in maxillary canine region of male beagle dogs, which involved the removal of the buccal side and near the side alveolar bone and periodontal membrane and smoothing root surfaces. We used fiber protein gel as a scaffold material, and compound r-PDLSCs and a-PDLSCs, respectively. transplanted them into the defect area. Finally dual-source CT was observed about alveolar bone regeneration. 5 weeks after surgery, r-PDLSCs and a-PDLSCs showed varying degrees of repair and regeneration in periodontal defects, while almost nothing in control group. The height of alveolar bone regeneration was significantly different in r-PDLSCs and a-PDLSCs. r-PDLSCs group restored an alveolar ridge height of about 30%, while the a-PDLSCs group restored an alveolar ridge height of about 50%. New bone was formed at defects edges and the middle part with a lower density. New bone fusion was observed between the edge region and the normal bone.After 10 weeks, r-PDLSCs and a-PDLSCs group both showed better restore ability with a higher alveolar ridge height than those in 5 weeks, r-PDLSCs group up to 60% and a-PDLSCs group almost 100%. The bone density was closer to normal level.This experiment shows that, in situ autogenous both r-PDLSCs and a-PDLSCs had repair ability of periodontal defects. But at the same time, a-PDLSCs showed a stronger regenerative ability.Through this study, autologous a-PDLSCs is expected to be the seed cell for periodontal tissue engineering in further clinical work, it can achieve the complete repair of periodontal tissue due to periodontal disease, and may be a new choice of optimization of seed cell. |
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